Preparation of Affinity Membrane for Protein Recovery

蛋白质回收亲和膜的制备

基本信息

批准号：
03650758
负责人：
SAITO Kyoichi
金额：
$ 1.09万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

A porous hollow-fibre membrane containing L-phenylalanine as a pseudo-biospecific ligand has been prepared by radiation induced grafting of glycidyl methacrylate onto polyethylene microfiltration hollow fibre, followed by coupling of the produced epoxide group with L-phenylalanine. The remaining epoxide group was hydrolysed into a diol group with sulphuric acid. The L-phenylalanine and the diol group acted in a complementary way as a pseudo-biospecific ligand and a hydrophilic group, respectively. The adsorption capacity of bovine gamma globulin on the resulting porous adsorbent could be determined by the specific surface area of the adsorbent when the ligand was in excess over the protein.A hollow-fibre affinity membrane containing hydrophobic amino acids as ligands was prepared by the radiation-induced grafting of glycidyl methacrylate onto a porous polyethylene hollow fibre and subsequent coupling with phenylalanine (Phe) or tryptophan (Trp). The densities of the Phe and Trp ligand of the resulting affinity membrane were 0.4 and 0.4 mol/kg, respectively. The Trp-containing affinity membrane exhibited a higher amount of adsorbed bovine y-globulin (BGG) than the Phe-containing membrane. To evaluate the adsorption behavior of the membrane, the BGG-containing buffer solution was permeated from the inside to the outside of the Trp-containing hollow-fibre affinity membrane through the ligand-immobilized pores. The breakthrough curves as a function of effluent volume coincided irrespective of the flow-rate, i.e. the residence time (55-220 s) of the solution across the membrane (thickness 0.83 mm), as a result of negligible mass transfer resistance. A series of chromatographic procedures, (adsorption-washing-elution) was repeated twice and a satisfactory quantitative elution was attained. The reproducible profile of the flux and the protein concentration assured a quantitative cycle of chromatography using the affinity membrane containing Trp as a ligand.

通过辐射诱导的糖基甲基丙烯酸甲酯接枝到聚乙烯微滤空心纤维上的辐射诱导接枝，将含有L-苯丙氨酸作为伪生物特异性配体的多孔空心纤维膜，随后由生产的epoxide组与L-苯基丙氨酸相结合。将剩余的环氧基团水解为含有硫酸的二醇基。 L-苯基丙氨酸和DIOL组分别以互补的方式作用，分别作为伪生物特异性配体和亲水性群。 The adsorption capacity of bovine gamma globulin on the resulting porous adsorbent could be determined by the specific surface area of the adsorbent when the ligand was in excess over the protein.A hollow-fibre affinity membrane containing hydrophobic amino acids as ligands was prepared by the radiation-induced grafting of glycidyl methacrylate onto a porous polyethylene hollow fibre and随后与苯丙氨酸（PHE）或色氨酸（TRP）耦合。所得亲和力膜的PHE和TRP配体的密度分别为0.4和0.4 mol/kg。含TRP的亲和力膜比含PHE的膜表现出更高量的吸附牛Y-球蛋白（BGG）。为了评估膜的吸附行为，含有BGG的缓冲液从内部到含TRP的空心纤维亲和力膜的内部渗透到通过配体 - 毫米毫米化的孔中渗透。突破性曲线是废水体积的函数，无论流量速率，即跨膜的溶液的停留时间（55-220 s），这是由于质量传播电阻而导致的。两次重复一系列色谱程序（吸附洗液），并获得了令人满意的定量洗脱。通量的可再现特征和蛋白质浓度使用包含TRP作为配体的亲和力膜的色谱法确保了定量循环。